'Up with the LRRK': a phosphorylated Rab10 assay for evaluation of LRRK2 activity and inhibitor engagement

Protein kinases catalyse the addition of phosphate groups to Ser/Thr and Tyr residues in cognate substrates and are mutated or hyperactive in a variety of diseases, making them important targets for rationally designed drugs. A good example is the Parkinson's disease-associated kinase, leucine-rich repeat kinase 2 (LRRK2), which is mutated (and probably hyperactive) in a small, but significant, subset of patients. An exciting new approach for personalised therapy is the development of central nervous system (CNS)-active small-molecule kinase inhibitors, which could be employed to ‘normalise’ LRRK2 signalling in affected cell types. However, the development of such drugs requires validated assays for the analysis of target engagement and the assembly of a set of tools for interrogating LRRK2, and its substrates, both in vitro and in vivo. A new study published in the Biochemical Journal by Ito et al. establishes that a ‘Phos-tag’™-binding assay can be exploited to measure phosphorylation of a recently identified LRRK2 substrate (Ras-related protein in brain 10 (Rab10)), and to compare and contrast relative catalytic output from disease-associated LRRK2 mutants. Powerful in vivo chemical genetic approaches are also disclosed, in which the catalytic activity of LRRK2 is unequivocally linked to the extent of Rab10 phosphorylation and the effects of chemically distinct LRRK2 inhibitors are matched with on-target inhibition mechanisms mediated through LRRK2 and its substrate Rab10. These important findings should simplify the generic analysis of Rab10 phosphorylation in model biological systems and are likely to be applicable to other substrates of LRRK2 (or indeed other kinases) for which phospho-specific antibodies are either absent or unsatisfactory

Rheostat-ing Mitosis

Ark1, the unique Aurora kinase in Schizosaccharomyces pombe, regulates multiple aspects of mitosis. In this issue of Chemistry & Biology, Kawashima and colleagues report the discovery and validation of a fungal Ark1 inhibitor, which they employ to evaluate the mitotic outputs of endogenous Ark1 signaling

Back to the future: new target-validated Rab antibodies for evaluating LRRK2 signalling in cell biology and Parkinson's disease

The addition of phosphate groups to substrates allows protein kinases to regulate a myriad of biological processes, and contextual analysis of protein-bound phosphate is important for understanding how kinases contribute to physiology and disease. Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase linked to familial and sporadic cases of Parkinson's disease (PD). Recent work established that multiple Rab GTPases are physiological substrates of LRRK2, with Rab10 in particular emerging as a human substrate whose site-specific phosphorylation mirrors hyperactive LRRK2 lesions associated with PD. However, current assays to quantify Rab10 phosphorylation are expensive, time-consuming and technically challenging. In back-to-back studies reported in the Biochemical Journal, Alessi and colleagues teamed up with clinical colleagues and collaborators at the Michael J. Fox Foundation (MJFF) for Parkinson's research to develop, and validate, a panel of exquisitely sensitive phospho-specific Rab antibodies. Of particular interest, the monoclonal antibody-designated MJFF-pRAB10 detects phosphorylated Rab 10 on Thr73 in a variety of cells, brain extracts, PD-derived samples and human neutrophils, the latter representing a previously unrecognised biological resource for LRRK2 signalling analysis. In the future, these antibodies could become universal resources in the fight to understand and quantify connections between LRRK2 and Rab proteins, including those associated with clinical PD.</jats:p

The evolving world of pseudoenzymes: proteins, prejudice and zombies

Pseudoenzymes are catalytically deficient variants of enzymes that are represented in all major enzyme families. Their regulatory functions in signalling pathways are shedding new light on the non-catalytic functions of active enzymes, and are suggesting new ways to target cellular signalling mechanisms with drugs

Rigorous Determination of the Stoichiometry of Protein Phosphorylation Using Mass Spectrometry

Quantification of the stoichiometry of phosphorylation is usually achieved using a mixture of phosphatase treatment and differential isotopic labeling. Here, we introduce a new approach to the concomitant determination of absolute protein concentration and the stoichiometry of phosphorylation at predefined sites. The method exploits QconCAT to quantify levels of phosphorylated and nonphosphorylated peptide sequences in a phosphoprotein. The nonphosphorylated sequence is used to determine the absolute protein quantity and serves as a reference to calculate the extent of phosphorylation at the second peptide. Thus, the stoichiometry of phosphorylation and the absolute protein concentration can be determined accurately in a single experiment

Bio-Zombie: the rise of pseudoenzymes in biology

Pseudoenzymes are catalytically dead counterparts of enzymes. Despite their first description some 50 years ago, the importance and functional diversity of these ‘fit-for-purpose’ polypeptides is only now being appreciated. Pseudoenzymes have been identified throughout all the kingdoms of life and, owing to predicted deficits in enzyme activity due to the absence of catalytic residues, have been variously referred to as pseudoenzymes, non-enzymes, dead enzymes, prozymes or ‘zombie’ proteins. An important goal of the recent Biochemical Society Pseudoenzymes-focused meeting was to explore the functional and evolutionary diversity of pseudoenzymes and to begin to evaluate their functions in biology, including cell signalling and metabolism. Here, we summarise the impressive breadth of enzyme classes that are known to have pseudoenzyme counterparts and present examples of known cellular functions. We predict that the next decades will represent golden years for the analysis of pseudoenzymes.</jats:p

Spindle checkpoint proteins Mad1 and Mad2 are required for cytostatic factor–mediated metaphase arrest

In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase. The budding uninhibited by benzimidazole (Bub) 1, mitotic arrest-deficient (Mad) 1, and Mad2 proteins promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to a cytoplasmic activity termed cytostatic factor (CSF), which appears not to be regulated by spindle dynamics. Here, we show that microinjection of Mad1 or Mad2 protein into early Xenopus laevis embryos causes metaphase arrest like that caused by Mos. Microinjection of antibodies to either Mad1 or Mad2 into maturing oocytes blocks the establishment of CSF arrest in meiosis II, and immunodepletion of either protein blocked the establishment of CSF arrest by Mos in egg extracts. A Mad2 mutant unable to oligomerize (Mad2 R133A) did not cause cell cycle arrest in blastomeres or in egg extracts. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2 or Bub1. These results suggest a model in which CSF arrest by Mos is mediated by the Mad1 and Mad2 proteins in a manner distinct from the spindle checkpoint

cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry

cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R(2)C(2) holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling